Fiona M. Tomley

Sequence of the gene encoding an immunodominant microneme protein of Eimeria tenella

A heterodisperse family of antigens, previously detected on sporozoites and merozoites of Eimeria tenella, has been localised to the microneme organelles within the sporozoite. Sequencing of genomic and cDNA clones shows that the gene for this antigen family contains 4 exons separated by 3 short (519, 226 and 156 nucleotides) intervening sequences and that the predicted polypeptide from the longest open reading frame has 4 structural domains. One of these contains 5 copies of the thrombospondin-like motif, previously identified in the partial sequence of the gene, which is conserved in a variety of molecules which have been demonstrated to have adhesive properties. A second domain of the polypeptide has strong similarity to a conserved region that occurs in another group of molecules which have adhesive properties, including the alpha subunits of several integrins, complement factor Bb and a number of extracellular matrix glycoproteins. Overall the antigen resembles the thrombospondin-related anonymous protein identified in the erythrocytic stage of Plasmodium falciparum. The structure of the gene supports a role for this microneme antigen in cell-cell or cell-matrix interactions.

Securing poultry production from the ever-present Eimeria challenge

The intestinal disease coccidiosis, caused by protozoan parasites of the genus Eimeria, is one of the most important livestock diseases in the world. It has a high impact in the poultry industry where parasite transmission is favoured by high-density housing of large numbers of susceptible birds. Coccidiosis control in poultry is achieved by careful husbandry combined with in-feed anticoccidial drugs or vaccination with live parasites. However, outbreaks of coccidiosis still occur and subclinical infections, which significantly impact on productivity and food security, are common due to widespread drug resistance, high parasite prevalence, and environmental persistence. Herein, we review some recent approaches for the production of cheaper third generation vaccines, based on robust methods for identification of immunoprotective antigens and the use of transgenic Eimeria.

The proteome of Toxoplasma gondii: integration with the genome provides novel insights into gene expression and annotation

BackgroundAlthough the genomes of many of the most important human and animal pathogens have now been sequenced, our understanding of the actual proteins expressed by these genomes and how well they predict protein sequence and expression is still deficient. We have used three complementary approaches (two-dimensional electrophoresis, gel-liquid chromatography linked tandem mass spectrometry and MudPIT) to analyze the proteome of Toxoplasma gondii, a parasite of medical and veterinary significance, and have developed a public repository for these data within ToxoDB, making for the first time proteomics data an integral part of this key genome resource.ResultsThe draft genome for Toxoplasma predicts around 8,000 genes with varying degrees of confidence. Our data demonstrate how proteomics can inform these predictions and help discover new genes. We have identified nearly one-third (2,252) of all the predicted proteins, with 2,477 intron-spanning peptides providing supporting evidence for correct splice site annotation. Functional predictions for each protein and key pathways were determined from the proteome. Importantly, we show evidence for many proteins that match alternative gene models, or previously unpredicted genes. For example, approximately 15% of peptides matched more convincingly to alternative gene models. We also compared our data with existing transcriptional data in which we highlight apparent discrepancies between gene transcription and protein expression.ConclusionOur data demonstrate the importance of protein data in expression profiling experiments and highlight the necessity of integrating proteomic with genomic data so that iterative refinements of both annotation and expression models are possible.

PCR identification of chicken Eimeria: A simplified read-out

A polymerase chain reaction (PCR) assay, based on the amplification of internal transcribed spacer 1 (ITS1) regions of ribosomal DNA, was developed for the chicken coccidian species Eimeria maxima, E. mitis and E. praecox. Thus, taking into account our previous work, a complete set of ITS1-based, species-specific primers for the detection and discrimination of all seven Eimeria species that infect the domestic fowl is now available. ITS1 primers for each of these seven species of Eimeria were also used as capture probes in a paper chromatography assay (PACHA). The addition of PACHA to the PCR assay provided a faster, more simplified read-out compared to staining of amplified bands in an agarose gel with ethidium bromide.

A Selective Review of Advances in Coccidiosis Research

Coccidiosis is a widespread and economically significant disease of livestock caused by protozoan parasites of the genus Eimeria. This disease is worldwide in occurrence and costs the animal agricultural industry many millions of dollars to control. In recent years, the modern tools of molecular biology, biochemistry, cell biology and immunology have been used to expand greatly our knowledge of these parasites and the disease they cause. Such studies are essential if we are to develop new means for the control of coccidiosis. In this chapter, selective aspects of the biology of these organisms, with emphasis on recent research in poultry, are reviewed. Topics considered include taxonomy, systematics, genetics, genomics, transcriptomics, proteomics, transfection, oocyst biogenesis, host cell invasion, immunobiology, diagnostics and control.

Induction of secretion and surface capping of microneme proteins in Eimeria tenella.

Micronemes are secretory organelles of the invasive stages of apicomplexan parasites and contain proteins that are important for parasite motility and host cell invasion. We have examined the induction of microneme secretion in the coccidian Eimeria tenella. When sporozoites were added to MDBK cells in culture, microneme proteins were secreted, capped backwards over the parasite surface and deposited onto underlying host cells from the posterior end of gliding parasites. Induction of secretion was also achieved by the addition of foetal calf serum, or purified albumin, to extracellular sporozoites. Microneme secretion per se was not dependent on parasites being able to move or to invade host cells. However, in the presence of cytochalasin D, which disrupts actin polymerisation and prevents parasite movement, microneme proteins were secreted from the apical tip but were not capped backwards over the sporozoite surface. These observations support the hypothesis that microneme proteins function as ligands which, when secreted out onto the parasite surface, form a link, either directly or indirectly, between the sub-pellicular actin myosin cytoskeletal motor of the parasite and the surface of target host cells.

Genomic analysis of the causative agents of coccidiosis in domestic chickens

Global production of chickens has trebled in the past two decades and they are now the most important source of dietary animal protein worldwide. Chickens are subject to many infectious diseases that reduce their performance and productivity. Coccidiosis, caused by apicomplexan protozoa of the genus Eimeria, is one of the most important poultry diseases. Understanding the biology of Eimeria parasites underpins development of new drugs and vaccines needed to improve global food security. We have produced annotated genome sequences of all seven species of Eimeria that infect domestic chickens, which reveal the full extent of previously described repeat-rich and repeat-poor regions and show that these parasites possess the most repeat-rich proteomes ever described. Furthermore, while no other apicomplexan has been found to possess retrotransposons, Eimeria is home to a family of chromoviruses. Analysis of Eimeria genes involved in basic biology and host-parasite interaction highlights adaptations to a relatively simple developmental life cycle and a complex array of co-expressed surface proteins involved in host cell binding.

MORN1 has a conserved role in asexual and sexual development across the apicomplexa.

ABSTRACT The gene encoding the membrane occupation and recognition nexus protein MORN1 is conserved across the Apicomplexa. In Toxoplasma gondii, MORN1 is associated with the spindle poles, the anterior and posterior rings of the inner membrane complex (IMC). The present study examines the localization of MORN1 during the coccidian development of T. gondii and three Eimeria species (in the definitive host) and erythrocytic schizogony of Plasmodium falciparum. During asexual proliferation, MORN1 is associated with the posterior ring of the IMCs of the multiple daughters forming during T. gondii endopolygeny and schizogony in Eimeria and P. falciparum. Furthermore, the expression of P. falciparum MORN1 protein peaked in late schizogony. These data fit a model with a conserved role for MORN1 during IMC assembly in all variations of asexual development. An important new observation is the reactivity of MORN1 antibody with certain sexual stages in T. gondii and Eimeria species. Here MORN1 is organized as a ring-like structure where the microgametes bud from the microgametocyte while in mature microgametes it is present near the flagellar basal bodies and mitochondrion. These observations suggest a conserved role for MORN1 in both asexual and sexual development across the Apicomplexa.

Characterisation of Plasmodium invasive organelles; an ookinete microneme proteome

Secretion of microneme proteins is essential to Plasmodium invasion but the molecular composition of these secretory organelles remains poorly defined. Here, we describe the first Plasmodium microneme proteome. Purification of micronemes by subcellular fractionation from cultured ookinetes was confirmed by enrichment of known micronemal proteins and electron microscopy. Quantitation of electron micrographs showed >14‐fold microneme enrichment compared to the intact ookinete, such that micronemes comprised 85% of the identifiable organelles in the fraction. Gel LC‐MS/MS of the most abundant protein constituents of the fraction identified three known micronemal proteins chitinase, CTRP, SOAP, together with protein disulphide isomerase (PDI) and HSP70. Highly sensitive MudPIT shotgun proteomics described a total of 345 proteins in the fraction. M1 aminopeptidase and PDI, the former a recognised target of drug development, were both shown to have a micronemal location by IFA. We further identified numerous proteins with established vesicle trafficking and signaling functions consistent with micronemes being part of a regulated secretory pathway. Previously uncharacterised proteins comprise the largest functional group of the microneme proteome and will include secreted proteins important to invasion.

Proteomic comparison of four Eimeria tenella life cycle stages: unsporulated oocyst, sporulated oocyst, sporozoite and second- generation merozoite

We report the proteomes of four life‐cycle stages of the Apicomplexan parasite Eimeria tenella. A total of 1868 proteins were identified, with 630, 699, 845 and 1532 found in early oocysts (unsporulated), late oocysts (sporulated), sporozoites and second‐generation merozoites, respectively. A multidimensional protein identification technology shotgun approach identified 812 sporozoites, 1528 merozoites and all of the oocyst proteins, whereas 2‐D gel proteomics identified 230 sporozoites and 98 merozoite proteins. Comparing the invasive stages, we find moving junction components RON2 in both, whereas AMA‐1 and RON4 are found only in merozoites and AMA‐2 and RON5 are only found in sporozoites, suggesting stage‐specific moving junction proteins. During early oocyst to sporozoite development, refractile body and most “glideosome” proteins are found throughout, whereas microneme and most rhoptry proteins are only found after sporulation. Quantitative analysis indicates glycolysis and gluconeogenesis are the most abundant metabolic groups detected in all stages. The mannitol cycle “off shoot” of glycolysis was not detected in merozoites but was well represented in the other stages. However, in merozoites we find more protein associated with oxidative phosphorylation, suggesting a metabolic shift mobilising greater energy production. We find a greater abundance of protein linked to transcription, protein synthesis and cell cycle in merozoites than in sporozoites, which may be residual protein from the preceding massive replication during schizogony.